1. Field of the Invention
The present invention relates generally to methods and compositions for detecting soluble .beta.-amyloid peptide (.beta.AP) in fluid samples. More particularly, the present invention relates to screening methods for the identification of inhibitors of .beta.AP production where .beta.AP is detected in vitro or in vivo and to diagnostic methods where .beta.AP is detected in patient samples.
Alzheimer's Disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms and course is currently known.
The brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Smaller numbers of these lesions in a more restricted anatomical distribution are also found in the brains of most aged humans who do not have clinical AD. Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D). At present, a definitive diagnosis of AD usually requires observing the aforementioned lesions in the brain tissue of patients who have died with the disease or, rarely, in small biopsied samples of brain tissue taken during an invasive neurosurgical procedure.
The principal chemical constituent of the amyloid plaques and vascular amyloid deposits (amyloid angiopathy) characteristic of AD and the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 39-43 amino acids designated the .beta.-amyloid peptide (.beta.AP) or sometimes A.beta., A.beta.P or .beta./A4. .beta.AP was first purified and a partial amino acid sequence reported in Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120:885-890. The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829.
Molecular biological and protein chemical analyses conducted during the last six years have shown that .beta.AP is a small fragment of a much larger precursor protein, referred to as the .beta.-amyloid precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans. Knowledge of the structure of the gene encoding APP has demonstrated that .beta.AP arises as a peptide fragment that is cleaved from APP by as-yet-unknown enzymes (proteases). The precise biochemical mechanism by which the .beta.AP fragment is cleaved from APP and subsequently deposited as amyloid plaques in the cerebral tissue and in the walls of cerebral and meningeal blood vessels is currently unknown.
Several lines of evidence indicate that progressive cerebral deposition of .beta.AP plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades (for review, see Selkoe (1991) Neuron 6:487). The single most important line of evidence is the discovery in 1991 that missense DNA mutations at amino acid 717 of the 770-amino acid isoform of APP can be found in affected members but not unaffected members of several families with a genetically determined (familial) form of AD (Goate et al. (1991) Nature 349:704-706; Chartier Harlan et al. (1991) Nature 353:844-846; and Murrell et al. (1991) Science 254:97-99) and is referred to as the Swedish variant. A double mutation changing lysine.sup.595 -methionine.sup.596 to asparagine.sup.595 -leucine.sup.596 (with reference to the 695 isoform) found in a Swedish family was reported in 1992 (Mullan et al. (1992) Nature Genet 1:345-347). Genetic linkage analyses have demonstrated that these mutations, as well as certain other mutations in the APP gene, are the specific molecular cause of AD in the affected members of such families. In addition, a mutation at amino acid 693 of the 770-amino acid isoform of APP has been identified as the cause of the .beta.AP deposition disease, HCHWA-D, and a change from alanine to glycine at amino acid 692 appears to cause a phenotype that resembles AD in some patients but HCHWA-D in others. The discovery of these and other mutations in APP in genetically based cases of AD proves that alteration of APP and subsequent deposition of its .beta.AP fragment can cause AD.
Despite the progress which has been made in understanding the underlying mechanisms of AD and other .beta.AP-related diseases, there remains a need to develop methods and compositions for diagnosis and treatment of the disease(s). Treatment methods could advantageously be based on drugs which are capable of inhibiting the generation of .beta.AP in vivo. To identify such drugs, it would be desirable to provide screening assays for potential drugs which can inhibit .beta.AP generation in in vivo and in vitro models. It would be further desirable to provide methods and compositions for diagnosis of .beta.AP-related conditions, where the diagnosis is based on detection of .beta.AP in patient fluid samples. Specific assays for .beta.AP detection should be capable of detecting .beta.AP in fluid samples at very low concentrations as well as distinguishing between .beta.AP and other fragments of APP which may be present in the sample.
2. Description of the Background Art
Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120:885-890 and U.S. Pat. No. 4,666,829, are discussed above. The '829 patent suggests the use of an antibody to the 28 amino acid .beta.AP fragment to detect "Alzheimer's Amyloid Polypeptide" in a patient sample and diagnose AD. No data demonstrating detection or diagnosis are presented.
Numerous biochemical electron microscopic and immunochemical studies have reported that .beta.AP is highly insoluble in physiologic solutions at normal pH. See, for example, Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 122:1131-1135; Masters et al. (1985) Proc. Natl. Acad. Sci. USA 82:4245-4249; Selkoe et al. (1986) J. Neurochem. 46:1820-1834; Joachim et al. (1988) Brain Research 474:100-111; Hilbich et al. (1991) J. Mol. Biol. 218:149-163; Barrow and Zagorski (1991) Science 253:179-182; and Burdick et al. (1992) J. Biol. Chem. 267:546-554. Furthermore, this insolubility was predicted by and is consistent with the amino acid sequence of .beta.AP which includes a stretch of hydrophobic amino acids that constitutes part of the region that anchors the parent protein (APP) in the lipid membranes of cells. Hydrophobic, lipid-anchoring proteins such as .beta.AP are predicted to remain associated with cellular membranes or membrane fragments and thus not be present in physiologic extracellular fluids. The aforementioned studies and many others have reported the insolubility in physiologic solution of native .beta.AP purified from AD brain amyloid deposits or of synthetic peptides containing the .beta.AP sequence. The extraction of .beta.AP from cerebral amyloid deposits and its subsequent solubilization has required the use of strong, non-physiologic solvents and denaturants. Physiologic, buffered salt solutions that mimic the extracellular fluids of human tissues have uniformly failed to solubilize .beta.AP.
Separate attempts to detect APP or fragments thereof in plasma or CSF have also been undertaken. A large secreted fragment of APP that does not contain the intact .beta.AP region has been found in human cerebrospinal fluid (Palmert et al. (1989) Proc. Natl. Acad. Sci. USA 86:6338-6342; Weidemann et al. (1989) Cell 57:115-126; Henriksson et al. (1991) J. Neurochem. 56:1037-1042; and Palmert et al. (1990) Neurology 40:1028-1034); and plasma (Podlisny et al. (1990) Biochem. Biophys. Res. Commun. 167:1094-1101). The detection of fragments of the carboxy-terminal portion of APP in plasma has also been reported (Rumble et al. (1989) N. Engl. J. Med 320:1446-1452) as has the failure to detect such fragments (Schlossmacher et al. (1992) Neurobiol. Aging 13:421-434).
Despite the apparent insolubility of native and synthetic .beta.AP, it has been speculated that .beta.AP could occur in body fluids, such as cerebrospinal fluid (CSF) or plasma (Wong et al. (1985) Proc. Natl. Acad. Sci. USA 92:8729-8732; Selkoe (1986) Neurobiol. Aging 7:425-432; Pardridge et al. (1987) Biochem. Biophys. Res. Commun. 145:241-248; Joachim et al. (1989) Nature 341:226-230; Selkoe et al. (1989) Neurobiol. Aging 10:387-395).
Several attempts to measure .beta.AP in CSF and plasma have been reported by both radioimmunoassay methods (Pardridge et al. (1987) Biochem. Biophys. Res. Commun., supra, and WO90/12870 published Nov. 1, 1990) and sandwich ELISAs (Wisniewski in Alzheimer's Disease, eds. Becker and Giacobini, Taylor and Francas, N.Y. pg. 206, 1990; Kim and Wisniewski in Techniques in Diagnostic Pathology, eds. Bullock et al., Academic Press, Boston pg. 106; and WO90/12871 published Nov. 1, 1990). While these reports detected very low levels of .beta.AP immunoreactivity in bodily fluids, attempts to directly purify and characterize this immunoreactivity further and determine whether it represented .beta.AP were not pursued, and the efforts were abandoned. The possibility of .beta.AP production by cultured cells was neither considered nor demonstrated. Retrospectively, the inability to readily detect .beta.AP in bodily fluids was likely due to the presence of amyloid precursor fragments with overlapping regions or fragments of .beta.AP that obscured measurements and to the lack of antibodies completely specific for intact .beta.AP. In fact, the previous findings by both Pardridge et al. and Kim et al. reported levels of .beta.AP four-to-fivefold lower than that shown in the present invention. This is presumably because the antibodies used by both groups would crossreact with other APP fragments containing part of .beta.AP known to be present in CSF thereby interfering with the measurement, if any, of intact .beta.AP. The present invention overcomes these difficulties with the use of monoclonal antibodies specific to an epitope in the central junction region of intact .beta.AP.
EP 444,856 provides a means of diagnosing Alzheimer's disease using a sandwich immunoassay to "Alzheimer's Disease Associated Protein" (ADAP). ADAP is defined as a material reactive with the monoclonal antibody termed Alz50, originally described by Wolozin et al. (1986) Science 232:648-650. Alz50 has more recently been shown to react specifically with phosphorylated forms of tau (Ksiezak-Reder et al. (1988) J. Biol. Chem. 263:7943-7947; Ueda et al. (1990) J. Neuroscience 10:3295-3304; Lee et al. (1991) Science 251:675-678). Hence, ADAPs represent phosphorylated forms of tau and are unrelated to the amyloid precursor protein of .beta.AP described in this invention.